The ability to determine the sequence of a polynucleotide is of great scientific importance. For example, the Human Genome Project is an ambitious international effort to map and sequence the three billion bases of DNA encoded in the human genome. When complete, the resulting sequence database will be a tool of unparalleled power for biomedical research. The major obstacle to the successful completion of this project concerns the technology used in the sequencing process.
The principle method in general use for large-scale DNA sequencing is the chain termination method. This method was first developed by Sanger and Coulson (Sanger et al., Proc. Natl. Acad. Sci. USA, 1977; 74: 5463-5467), and relies on the use of dideoxy derivatives of the four nucleoside triphosphates which are incorporated into a nascent polynucleotide chain in a polymerase reaction. Upon incorporation, the dideoxy derivatives terminate the polymerase reaction and the products are then separated by gel electrophoresis and analysed to reveal the position at which the particular dideoxy derivative was incorporated into the chain.
Although this method is widely used and produces reliable results, it is recognised that it is slow, labour-intensive and expensive.
An alternative sequencing method is proposed in EP-A-0471732, which uses spectroscopic means to detect the incorporation of a nucleotide into a nascent polynucleotide strand complementary to a target. The method relies on an immobilised complex of template and primer, which is exposed to a flow containing only one of the different nucleotides. Spectroscopic techniques are then used to measure a time-dependent signal arising from the polymerase catalysed growth of the template copy. The spectroscopic techniques described are surface plasmon resonance (SPR) spectroscopy, which measures changes in an analyte within an evanescent wave field, and fluorescence measuring techniques. However, limitations of this method are recognised; the most serious for the SPR technique being that, as the size of the copy strand grows, the absolute size of the signal also grows due to the movement of the strand out of the evanescent wave field, making it harder to detect increments. The fluorescence measuring techniques have the disadvantage of increasing background interference from the fluorophores incorporated on the growing nascent polynucleotide chain. As the chain grows, the background “noise” increases and the time required to detect each nucleotide incorporation needs to be increased. This severely restricts the use of the method for sequencing large polynucleotides.
Single fragment polynucleotide sequencing approaches are outlined in WO-A-9924797 and WO-A-9833939, both of which employ fluorescent detection of single labelled nucleotide molecules. These single nucleotides are cleaved from the template polynucleotide, held in a flow by an optical trap (Jett, et al., J. Biomol. Struc. Dyn, 1989; 7:301-309), by the action of an exonuclease molecule. These cleaved nucleotides then flow downstream within a quartz flow cell, are subjected to laser excitation and then detected by a sensitive detection system. However, limitations of this method are recognised; the most serious for the exonuclease technique being the fact that the labelled nucleotides severely affect the processivity of the exonuclease enzyme. Other limitations of this method include ‘sticking’ of the nucleotide(s) to the biotin bead used to immobilise the polynucleotide fragment, thus resulting in the nucleotide flow becoming out of phase; inefficiency and length limitation of the initial enzymatic labelling process; and the excitation ‘cross-over’ between the four different dye molecules resulting in a greatly increased error rate.
There is therefore a need for an improved method, preferably at the single fragment level, for determining the sequence of polynucleotides, which significantly increases the rate and fragment size of the polynucleotide sequenced and which is preferably carried out by an automated process, reducing the complexity and cost associated with existing methods.